A major challenge of cancer therapy is preferential destruction of malignant cells with sparing of the normal tissue. Critical for successful eradication of malignant disease are early detection and selective ablation of the malignancy. This proposal addresses both issues.
Multiple, complementary techniques for tumor detection, including magnetic resonance, scintigraphic and optical imaging are under active development, each approach has particular strengths and advantages. Optical imaging includes measurement of absorption of endogenous molecules (e.g. hemoglobin) or administered dyes, detection of bioluminescence in preclinical models, and detection of fluorescence from endogenous fluorophores or from targeted exogenous molecules. Fluorescence, which involves absorption of light and re-emission at a longer wavelength, can be highly sensitive: a typical cyanine dye with a lifetime of 0.6 nsec can emit up to 1032 photons/second/mole. A sensitive optical detector can image <103 photons/second. Thus even with low excitation power, low concentrations of fluorescent molecular beacons can be detected.
As with other non-invasive techniques, fluorescence imaging has the potential for performing in vivo diagnosis in situ, with real time display of the resulting information [46]. Optical tomographic techniques are being devised to visualize the fluorescent probes within tissue volumes. Optical imaging instruments may be simpler and less expensive to operate than those required for other imaging technologies, permitting their eventual application by less specialized medical centers. Therapeutically in applications such as endoscopic examination, fluorescence imaging can allow precise assessment of the location and size of a tumor, and provide information on its invasiveness. During debulking surgery, where malignant loci can be difficult to identify, the presence of a fluorescent signal might assist the surgeon in identifying the diseased site.
The optimal wavelength range for in vivo fluorescence excitation and emission is determined by tissue optical properties. Hemoglobin has strong absorption at wavelengths less than about 600 nm and there can be significant background fluorescence from endogenous biomolecules up to about 680 nm. At longer wavelengths into the near infrared (NIR), tissue absorption and scattering decrease with wavelength. As shown in FIG. 1 there is a large increase in light penetration as wavelengths increase from ˜600 to 800 nm. In addition, the difference between the fluorophore's absorption and emission bands (i.e. its Stokes shift), should be at least 20 nm, to readily discriminate between the excitation and emission light. Many NIR fluorescent dyes are based on carbocyanine molecules such as indocyanine green (ICG), an FDA-approved agent with a 730 nm excitation and 830 nm emission maxima. Various novel ICG analogs have been evaluated because of the high biocompatibility and desirable spectral properties of the carbocyanines.
A challenge is to deliver the dyes selectively and in high enough concentration to detect small tumors. Use of ICG alone to image hypervascular or “leaky” angiogenic vessels around tumors has been disappointing, due to the dye's limited intrinsic tumor selectivity. Multiple approaches have been employed to improve optical probe localization, including administering it in a quenched form that is activated within tumors, or coupling the fluorescent agents to antibodies or small molecules such as receptor ligands. Recent studies have focused on developing dye conjugates of small bioactive molecules, to improve rapid diffusion to target tissue, incorporate combinatorial and high throughput strategies to identify and optimize new probes, and enhance in vivo stability of the compounds. Some of the peptide and folic-acid analogs of certain ICG derivatives have shown some tumor specificity and are at initial stages of pre-clinical studies.
Recently, a new class of multicarboxilate-containing carbocyanine probes have been reported for use as optical scaffolds that not only serve as fluorescent antennae but also participate in structural assembly of the multivalent molecular construct. The peripheral carboxylic acids that are distal to the chromophore core allow facile conjugation with biomolecules and retain the desirable NIR spectral properties of the dendritic molecule. However, none of these compounds are designed for both tumor detection and therapy. It is important to develop targeting strategies that cope with the heterogeneity of tumors in vivo, where there are inconsistent and varying expression of targeable sites. As discussed below,
Photodynamic therapy (PDT) is a clinically effective and still evolving locally selective therapy for cancers. PDT's utility has been demonstrated for varying photosensitizers and multiple types of disease. It is FDA approved for early and late stage lung cancer, obstructive esophageal cancer, high-grade dysplasia associated with Barrett's esophagus, age-related macular degeneration and actinic keratoses. PDT employs tumor localizing photosensitizers that produce reactive singlet oxygen upon absorption of light. Subsequent oxidation-reduction reactions also can produce superoxide anions, hydrogen peroxide and hydroxyl radicals. Photosensitizers have been designed which localize relatively specifically in certain subcellular structures such as the mitochondria, which are exquisitely sensitive targets. On the tumor tissue level, direct photodynamic tumor cell kill, destruction of the tumor supporting vasculature and possibly activation of the innate and adaptive anti-tumor immune system interact to destroy the malignant tissue. The preferential killing of the targeted cells (e.g. tumor), rather than adjacent normal tissues, is essential for PDT, and the preferential target damage achieved in clinical applications is a major driving force behind the use of the modality. The success of PDT relies on development of tumor-avid molecules that are preferentially retained in malignant cells but cleared from normal tissues.
Clinical PDT initially was developed at Roswell Park Cancer Institute which has one of the world's largest basic and clinical research programs. Initially the RPCI group developed Photofrin®, the first generation FDA approved hematoporphyrin-based compound. Subsequently, our group has investigated the structure activity relationships for tumor selectivity and photosensitizing efficacy, and used the information to design new photosensitizers with high selectivity and desirable pharmacokinetics. Although the mechanism of porphyrin retention by tumors in not well understood, the balance between lipophilicity and hydrophilicity is recognized as an important factor. In efforts to develop effective photosensitizers with the required photophysical characteristics, chlorophyll-a and bacteriochlorophyll-a were as the substrates. An extensive QSAR study on a series of the alkyl ether derivatives of pyropheophorbide-a (660 nm) led to selection of the best candidate, HPPH (hexyl ether derivative), [98, 99] currently in promising Phase II clinical trials. Our PS development currently is being extended in purpurinimide (700 nm) and bacteriopurpurinimde (780-800 nm) series with high singlet oxygen (1O2) producing capability. The long wavelength absorption is important for treating large deep-seated tumors, because it both increases light penetration and minimizes the number of optical fibers needed for light delivery within the tumor.
Some of these compounds are highly tumor avid. As shown in FIG. 18 in the Preliminary Data, with an optimized system, 48 and 72 h after administration, ratios of ˜6:1 and 10:1 between the tumor and surrounding muscle and other body sites have been achieved, except for the liver, spleen and kidney. This in vivo selectivity is 2-3 fold greater than that reported for a carbocyanine dye coupled to a somatostatin analog.
Photosensitizers (PS), especially tetrapyrollic photosensitizers such as porphyrins, are not optimal for tumor detection. Examples of such tetrapyrollic photosensitizers are intended to include modified chlorines, bacteriochlorins, hematoporphyrins, porphyrins, purpurins, purpurin imides, and pyropheophorbides. All of the foregoing are referred to herein as porphyrins. Examples of such photodynamic compounds are described in numerous patents in this area that have been applied for and granted world wide on these photodynamic compounds. Reference may be had, for example to the following U.S. patents which are incorporated herein by reference: U.S. Pat. Nos. 4,649,151; 4,866,168; 4,889,129; 4,932,934; 4,968,715; 5,002,962; 5,015,463; 5,028,621; 5,145,863; 5,198,460; 5,225,433; 5,314,905; 5,459,159; 5,498,710; and 5,591,847.
Such photosensitizers generally fluoresce and the fluorescence properties of these porphyrins in vivo has been exploited by several investigators for the detection of early-stage cancers in the lung, bladder and various other sites. In addition, for treatment of early disease or for deep seated tumors the fluorescence can be used to guide the activating light. However, such photosensitizers are not optimal fluorophores for tumor detection for several reasons: (i) They have low quantum yields. Because the excited state energy is transferred to the triplet state and then to molecular oxygen, efficient photosensitizers tend to have lower fluorescence efficiency (quantum yield) than compounds designed to be fluorophores, such as cyanine dyes. (ii) They have small Stokes shifts. Porphyrin-based photosensitizers have a relatively small difference between the long wavelength absorption band and the fluorescence wavelength (Stokes shift), which makes it technically difficult to separate the fluorescence from the excitation wavelength. (iii) They have relatively short fluorescent wavelengths, <800 nm, which are not optimal for deep tissue penetration.
Bifunctional photosensitizer-fluorophore conjugates can optimize tumor detection and treatment. Certain bifunctional conjugates have been recently developed that use tumor-avid photosensitizers to target the NIR fluorophores to the tumor. The function of the fluorophore is to visualize the tumor location and treatment site. The presence of the photosensitizer allows subsequent tumor ablation. A compound that effectively functions both as a fluorescence imaging agent and a photosensitizer would create an entirely new paradigm for tumor detection and therapy. The optical imaging allows the clinician performing photodynamic therapy to continuously acquire and display patient data in real-time. This “see and treat” approach may determine where to treat superficial carcinomas and how to reach deep-seated tumors in sites such as the breast with optical fibers delivering the photoactivating light.
Metallized photodynamic compounds have shown promise in in vivo PDT efficacy and fluorescence imaging potential. However, the therapeutic dose was significantly higher than the therapeutic dose and a considerable fluorescence resonance energy transfer (FRET) was observed between the two chromophores.